CN102213718A - Heat-shrinkable combined micro-channel chip, and preparation and application method - Google Patents

Heat-shrinkable combined micro-channel chip, and preparation and application method Download PDF

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CN102213718A
CN102213718A CN2011100721849A CN201110072184A CN102213718A CN 102213718 A CN102213718 A CN 102213718A CN 2011100721849 A CN2011100721849 A CN 2011100721849A CN 201110072184 A CN201110072184 A CN 201110072184A CN 102213718 A CN102213718 A CN 102213718A
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micro
step
channel chip
concentration
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向安
郭晏海
颜真
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中国人民解放军第四军医大学
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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/558Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/551Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
    • G01N33/552Glass or silica

Abstract

The invention relates to a heat-shrinkable combined micro-channel chip, and a preparation and application method. In a biochip technique, the hybridization in closed micro-channels such as a capillary and the like has the advantages of rapid, sensitive and specific hybridization reaction, but the fixing of the probe molecules in different space positions in the capillary has the disadvantages of high technical barriers and high cost and is not beneficial to the production in batches. In the invention, the principle that target molecules are fixed on the inner wall of a substrate slot and a memory material in an expanded state is shrunk at the temperature when crystals are fused is utilized and applied to the fields of nucleic acid detection, protein antigen and antibody detection and biological toxin detection. The preparation method provided by the invention is simple and rapid, is sensitive in the course of detection application, has the advantages of low cost and high flux and is extremely suitable for the detection of a biological macromolecule sample.

Description

热缩组合式微通道芯片、制备及应用方法 Micro-channel chip shrink composition, preparation and application methods

技术领域 FIELD

[0001 ] 本发明涉及一种热缩组合式微通道芯片、制备及应用方法。 [0001] The present invention relates to a micro-channel chip shrink composition, preparation and application methods. 背景技术 Background technique

[0002] 生物杂交技术是利用互补的核苷酸序列、蛋白抗原或抗体通过特异性的结合形成稳定的杂合分子,从而实现对靶分子的特异性检测。 [0002] Biological hybridization techniques using a nucleotide sequence complementary to the protein antigen or antibody to form a stable hybrid molecule by specific binding in order to achieve specific detection of target molecules. 而生物芯片技术是指在支持物上不同空间位置上固定各种特异的探针分子,然后与经标记的样品分子杂交,实际上就是一种大规模集成固相杂交。 And biochip technology refers to various specific fixed on the support different spatial locations on the probe molecule, and then hybridized with labeled sample molecule, it is actually a large scale integrated solid-phase hybridization. 毛细管等封闭式微通道内杂交具有杂交反应迅速、灵敏、特异的优势, 但是在毛细管内不同空间位置上的探针分子的固定存在较高的技术壁垒,且造价昂贵,不利于规模生产。 Enclosed within the capillary microchannel rapid hybridization with the hybridization reaction, sensitive and specific advantages, but there is a higher technical barriers in the capillary probe molecules fixed on the different spatial locations, and costly, it is not conducive to mass production.

发明内容 SUMMARY

[0003] 本发明的目的是提供一种热缩组合式微通道芯片、制备及应用方法,在基质槽内壁不同位置固定探针分子、利用扩张状态的记忆材料在结晶熔化温度时发生收缩的原理, 使之与基质槽紧密结合形成微通道芯片,并应用于核酸检测、蛋白抗原抗体检测以及环境生物毒素检测的领域。 [0003] The object of the present invention is to provide a micro-channel chip shrink composition, preparation and method of application, the probe molecules fixed in different positions a substrate tank inner wall, using the principles of expanded state of the memory material shrinkage occurs when the crystalline melting temperature, so closely integrated with the micro-channel chip groove forming the matrix, and used in nucleic acid detection, antibody detection of protein antigens and biological environmental endotoxin detection.

[0004] 本发明所采用的技术方案为: [0004] The technical proposal of the present invention are:

热缩组合式微通道芯片的制备方法,其特征在于: 由以下步骤实现: Microchannel heat shrinkable compositions chip production method, wherein: implemented by the following steps:

步骤一:将带有U型槽的玻璃基质置于体积浓度为6. 5%的酸溶液中,70°C水浴超声清洗1小时;再置于质量浓度为10%的碱溶液中,80°C水浴超声清洗1小时;超纯水清洗后,真空干燥30分钟; Step one: A glass substrate was placed with a U-shaped groove volume concentration of 6.5% of the acid solution, 70 ° C water bath for 1 hour ultrasonic cleaning; and placed in an alkali solution concentration of 10% by mass of, 80 ° C water bath for 1 hour ultrasonic cleaning; washing ultrapure water, and dried under vacuum for 30 min;

步骤二:对玻璃基质的U型槽内壁进行氨基化处理和醛基化处理; 步骤三:体积为0. 1 μ L、摩尔浓度为10 μ mol/L生物素标记的核酸探针溶液经毛细作用吸入内径为250 μ m玻璃点样管内,加至玻璃基质的U型槽内壁,室温避光静置M小时, 在质量浓度为0. 1%的十二烷基硫酸钠溶液中超声清洗5分钟,超纯水清洗3次,真空干燥30分钟,4°C保存; Step two: U-shaped groove on the inner wall of the glass substrate and the aldehyde amination treatment process; Step three: a volume of 0. 1 μ L, molar concentration of the nucleic acid probe 10 μ mol / L biotin-labeled solution was capillary suction effect of the inner diameter of 250 μ m spotted glass tube, were added to U-shaped groove of the inner wall of the glass matrix, m standing hours at room temperature in the dark, at the concentration of 0.1% sodium dodecyl sulfate solution was ultrasonically cleaned minutes, washed three times with ultrapure water, and dried under vacuum for 30 minutes, 4 ° C storage;

步骤四:将内径为3. 0 mm,热缩比为2 :1的铁氟龙透明高聚物热缩材料套管置于体积浓度为6. 5%的酸溶液中,超声清洗30分钟;再置于质量浓度为10%的碱溶液中,超声清洗30分钟,超纯水清洗;100 μ L无水乙醇来回穿梭冲洗30分钟,超纯水清洗;真空冷冻干燥, 洁净工作区保存,室温保存; Step four: an inner diameter of 3. 0 mm, heat shrink ratio of 2: Teflon sleeve thermal material transparent polymer 1 was placed in a concentration of 6.5% by volume of the acid solution, 30 minutes ultrasonic cleaning; and then placed in a 10% concentration of an alkali solution, ultrasonically cleaned for 30 minutes with ultrapure water; 100 μ L of absolute ethanol and fro rinsed for 30 minutes with ultrapure water; vacuum freeze dry, clean work areas, room temperature save;

步骤五:将U型槽内固定有核酸探针的玻璃基质放置于清洗后的热缩材料套管中, 80°C真空干燥箱内均勻受热5分钟,热缩后套管高度透明,与玻璃基质的U型槽结合紧密。 Step 5: U-shaped groove of a nucleic acid probe fixed to the glass substrate is placed in the heat-shrinkable sleeve material after cleaning, the heat evenly within 80 ° C vacuum oven for 5 minutes and highly transparent heat-shrinkable tubing, glass U-shaped groove substrate binding tightly.

[0005] 步骤一中的酸溶液为硝酸溶液,碱溶液为氢氧化钠溶液。 [0005] Step a solution of the acid is nitric acid solution, an alkaline solution is sodium hydroxide solution.

[0006] 步骤二中的氨基化处理由以下步骤实现:经步聚一处理后的带有U型槽的玻璃基质浸泡于体积比为1 :9的3-氨基丙基三甲氧基硅烷/甲苯溶液中,置于80°C水浴超声30分钟,重复4次,1000转/分钟水平式离心5分钟;依次用分析纯甲苯溶液、体积浓度为95% 的乙醇溶液超声10分钟; [0006] The amination process step two is realized by the steps of: a glass substrate with a U-shaped groove after processing by Buju immersed in a volume ratio of 1: 3-aminopropyl trimethoxysilane 9 / toluene solution and placed in an ultrasonic water bath at 80 ° C for 30 minutes was repeated four times, 1000 rev / min horizontal centrifuged for 5 minutes; AR toluene solution washed with a volume concentration of 95% ethanol solution was sonicated for 10 minutes;

步骤二中的醛基化处理由以下步骤实现:将经过氨基化处理过的带有U型槽的玻璃基质置于体积浓度为10%的戊二醛磷酸缓冲液中,室温放置M小时,纯净水内超声清洗5分钟,真空干燥。 In step dialdehyde processing is implemented by the steps of: after amination treated glass substrate was placed with a U-shaped groove of a volume concentration of 10% glutaraldehyde phosphate buffer, incubated at room temperature M h, pure the water ultrasonic cleaning for 5 minutes, and dried in vacuo.

[0007] 步骤四中的酸溶液为硝酸溶液,碱溶液为氢氧化钠溶液。 In [0007] Step tetracarboxylic acid solution is a nitric acid solution, an alkaline solution is sodium hydroxide solution.

[0008] 如所述的制备方法得到的热缩组合式微通道芯片。 [0008] The production method of the thermal composition obtained micro-channel chip.

[0009] 热缩组合式微通道芯片的应用方法,其特征在于: 由以下步骤实现: [0009] The method of application of micro-channel chip shrink composition, wherein: implemented by the following steps:

步骤一:芯片腔道内吸入15μ L的预杂交液,热缩组合式微通道芯片垂直固定于杂交仪内旋转杆上,在42°C、40转/分钟的条件下,预杂交液在微通道芯片腔道内穿梭20分钟, IOml超纯水以30ml/min流速冲洗微通道芯片腔道,1000转/分钟水平式离心3分钟; Step a: under the suction of 15μ L prehybridization solution, a combination of micro-channel chip shrink chip vertically fixed to the rotating rod lumen within the hybridizer at 42 ° C, 40 rev / min, prehybridization solution in a micro-channel chip the shuttle lumen 20 minutes, IOml ultrapure water at 30ml / min flow rate of irrigation micro-channel chip cavity 1000 rev / min horizontal centrifuge for three minutes;

步骤二:脱氧核苷酸序列靶分子与杂交溶液按1 :4体积比混合得到杂交反应液,杂交仪内,42Ό、15μ L杂交反应液、40转/分钟的条件下,在微通道芯片腔道内穿梭30分钟; Step two: a deoxynucleotide with the sequence of the target molecule by hybridization solution 1: 4 volume ratio under the conditions of a hybridization reaction solution obtained, the hybridizer, 42Ό, 15μ L hybridization reaction solution, 40 rev / min, in the micro-channel chip chamber Road shuttle within 30 minutes;

步骤三:55°C条件下,将5. Oml洗脱液1以lOml/min流速冲洗微通道芯片腔道,然后将IOml洗脱液2以lOml/min流速冲洗微通道芯片腔道; Step Three: 55 ° C for conditions 1 to 5. Oml eluent to lOml / min flow rate of irrigation micro-channel chip cavity, and then the eluant flushed IOml micro-channel chip cavity at lOml / min flow rate;

步骤四:芯片腔道内吸满由PH为7.0的磷酸盐缓冲液经1500倍稀释后的亲和链霉素-碱性磷酸酶,于37°C下避光放置20分钟,再由10ml、pH为7. O的磷酸盐缓冲液以IOml/ min流速冲洗芯片腔道; Step Four: suck chip cavity 1500-fold diluted by the PH 7.0 phosphate buffer by affinity streptomycin - alkaline phosphatase, placed in a dark 37 ° C and 20 minutes, then a 10ml, pH 7. O phosphate buffer at IOml / min flow rate of flush chip cavity;

步骤五:将25 μ L显色液加入微通道芯片腔道,37°C下避光放置20分钟; 步骤六:观察实验结果并拍照记录。 Step 5: 25 μ L were added to a color micro-channel chip cavity, 37 ° C protected from light for 20 min; Step Six: The results were observed and photographed record.

[0010] 所述的步骤一中的预杂交液成份为lOOug/ml鲑鱼精、质量浓度为0. 1%的十二烷基硫酸钠溶液、质量浓度为0. 1%牛血清白蛋白和5X柠檬酸钠缓冲液。 [0010] a step in the pre-hybridization solution composition was lOOug / ml salmon sperm, at a concentration of 0.1% by mass of sodium lauryl sulfate solution at the concentration of 0.1% bovine serum albumin and 5X sodium citrate buffer.

[0011] 所述的步骤二中的杂交溶液的成份为体积浓度为50%的去离子甲酰胺、5X柠檬酸钠缓冲液和质量浓度为0. 1%的十二烷基硫酸钠溶液。 [0011] The composition of the two step hybridization solution to a concentration of 50% by volume of deionized formamide, 5X and sodium citrate buffer at a concentration of 0.1% by mass of sodium lauryl sulfate solution.

[0012] 所述的步骤三中的洗脱液1,100 μ L体积的成份为20Χ柠檬酸钠缓冲液10 μ L、 质量浓度为10%的十二烷基硫酸钠溶液1 μ L和无菌超纯水89 μ L ;所述的洗脱液2,100 μ L 体积的成份为20 X柠檬酸钠缓冲液0. 5 μ L、质量浓度为10%的十二烷基硫酸钠溶液Iul和无菌超纯水98. 5 μ L。 [0012] Step Three in the eluate 1,100 μ L volume of ingredients for 20Χ sodium citrate buffer, 10 μ L, concentration of 10% by mass sodium lauryl sulfate solution and 1 μ L None bacteria ultrapure water 89 μ L; [mu] L of the eluate volume ingredients 2,100 20 X of sodium citrate buffer, 0. 5 μ L, concentration of 10% by mass sodium lauryl sulfate solution Iul and sterile ultra-pure water 98. 5 μ L.

[0013] 所述的步骤五中的显色液的成分为5-溴-4-氯-3-吲哚基-磷酸盐、氯化硝基四氮唑蓝和磷酸盐缓冲液,体积比为5 :10 :1500. Component [0013] The fifth step of the color developing solution of 5-bromo-4-chloro-3-indolyl - phosphate, nitro blue tetrazolium chloride and phosphate buffer, the volume ratio of 5:10: 1500.

[0014] 本发明具有以下优点: [0014] The present invention has the following advantages:

本发明所涉及制备方法简捷快速,检测应用时灵敏、成本低,且具有较高通量,十分适宜于生物大分样本的检测。 The present invention relates to the preparation of means for rapid, sensitive detection application, low cost, and has high throughput, is suitable for detecting a biological specimen Oita.

附图说明 BRIEF DESCRIPTION

[0015] 图1为热缩组合式微通道芯片的结构图。 [0015] FIG. 1 is a configuration diagram of a combination of micro-channel chip shrink.

[0016] 图2为热缩组合式微通道芯片上探针分子结合位置的示意图。 [0016] FIG. 2 is a schematic view of a combination of on-chip thermal probe molecule binding sites microchannels.

[0017] 图3为热缩组合式微通道芯片的化学显色结果示意图。 [0017] FIG. 3 is a schematic view of a chemical thermal coloration composition results in micro-channel chip.

5[0018] 图中,1-带有U型槽的玻璃基质,2-铁氟龙透明高聚物热缩材料套管,3-探针分子标记区,①〜②:阳性对照;③〜④:阴性对照;⑤〜⑦:野生型检测组;⑧〜⑩:突变型检测组。 5 [0018] FIG, 1 a glass substrate with a U-shaped groove, 2- transparent Teflon heat shrink polymeric material casing, 3- molecular probe mark area, ①~②: positive control; ③~ ④: negative control; ⑤~⑦: detecting the wild-type group; ⑧~⑩: mutant-type detection group.

具体实施方式 Detailed ways

[0019] 下面结合具体实施方式对本发明进行详细的说明。 [0019] The present invention will be described in detail with reference to specific embodiments.

[0020] 记忆效应是指辐射交联聚乙烯等结晶或非结晶聚合材料加热到熔点以上时,晶粒虽然溶化,但并不出现流动状态,而具有如橡胶一类物质的弹性。 [0020] Memory effect refers to radiation crosslinked polyethylene crystalline or amorphous polymeric material is heated above the melting point, melt while crystal grains, but does not appear to flow state, and a rubber having elasticity such substances. 若此时使聚乙烯扩张,那么冷却定型后仍能保持扩张状态;如果将这种扩张聚乙烯重新加热到结晶熔化温度,这种聚合物材料会“记忆”起其未扩张时原来的形态并重新收缩恢复原样。 At this time, if the expansion of the polyethylene, then after cooling and shaping can maintain the expanded state; if the original shape when reheated to such expandable polyethylene crystalline melting temperature, this polymer material "remembers" from its unexpanded and shrink again restored to their original. 高分子记忆材料就具有这种“形状记忆效应”的功能,是指交联的聚乙烯等结晶或非结晶聚合材料加热到熔点以上时,晶粒虽然溶化,但并不出现流动状态,而具有如橡胶一类物质的弹性,若此时使聚乙烯扩张,那么冷却定型后仍能保持扩张状态,如果将这种扩张聚乙烯重新加热到结晶熔化温度,这种聚合物材料会“记忆”起其未扩张时原来的形态并重新收缩恢复原样的功能。 Polymer memory material having such a function on the "shape memory effect" refers to the crosslinking of the polyethylene crystalline or amorphous polymeric material is heated to above the melting point, melt while crystal grains, but does not appear flowable state, and having a resilient rubber-based material, if at this time the expansion of the polyethylene, then after cooling and shaping can maintain the expanded state, if such expandable polyethylene reheated to a crystalline melting temperature, this polymer material "remembers" from the original shape and re-contraction restored to their original function when it is not expanded. 用高分子记忆材料制造的透明管,加热后管径会收缩,利用这一特性可用于管内器件的固定和密封。 A transparent tube for producing a polymer memory material, the diameter will shrink upon heating, use of this feature can be used for fixing the tube and the sealing device. 本发明利用高分子记忆材料透明管的热缩功能,将进行了多点标记的玻璃基质进行热缩合,以得到具有较高检测通量的微通道生物芯片。 The present invention features the use of heat-shrinkable polymer memory material transparent tube, a multi-point will be marked glass substrate is subjected to thermal condensation to obtain a micro-channel biochip having a higher detection throughput.

[0021] 本发明所涉及的一种热缩组合式微通道芯片的制备方法如下: 1、带有U型槽的玻璃基质的前处理: [0021] A heat shrinkable compositions according to the present invention a method for preparing micro-channel chip is as follows: 1, a glass substrate pre-treatment with U-groove:

将长5. 0cm,U型槽内径1. 8mm,外径2. Omm的U型槽玻璃基质置于体积浓度为6. 5%的硝酸溶液中,70°C水浴超声清洗1小时;再置于质量浓度为10%的氢氧化钠溶液中,80°C水浴超声清洗1小时;超纯水清洗后,真空干燥30分钟。 Long 5. 0cm, U-groove inner diameter of 1. 8mm, an outer diameter of 2. Omm U-shaped groove placed in the glass matrix at a concentration of 6.5% by volume of nitric acid solution, 70 ° C water bath for 1 hour ultrasonic cleaning; then set the mass concentration of 10% sodium hydroxide solution, 80 ° C water bath for 1 hour ultrasonic cleaning; washing ultrapure water, and dried in vacuo for 30 minutes. 前处理的目的是为了对U型槽进行彻底清理,去除U型槽中的尘埃等物质。 Purpose of the pretreatment is to thoroughly clean the U-shaped grooves, to remove dust and other substances in the U-shaped groove.

[0022] 2、玻璃基质U型槽内壁的化学基团修饰: (a)玻璃基质U型槽内表面的氨基化处理: [0022] 2, the inner wall of the glass substrate of the U-shaped groove chemical group modification: (a) amino process U-groove surface of the glass substrate:

经步聚1处理后的玻璃基质U型槽浸泡于体积比为1 :9的3-氨基丙基三甲氧基硅烷/甲苯溶液中,置于80°C水浴超声30分钟,重复4次,1000转/分钟水平式离心5分钟;依次用分析纯甲苯溶液、体积浓度为95%的乙醇溶液超声10分钟。 U-shaped groove through the glass substrate 1 after processing Buju immersed in a volume ratio of 1: 3-aminopropyl trimethoxysilane / toluene solution 9, placed in an ultrasonic water bath at 80 ° C for 30 minutes was repeated four times, 1000 rev / min horizontal centrifuged for 5 minutes; AR toluene solution washed with a volume concentration of 95% ethanol solution was sonicated for 10 minutes.

[0023] ( b )玻璃基质U型槽内表面的醛基化处理: [0023] (b) an aldehyde of the U-shaped groove treated glass substrate surface:

将带有U型槽的玻璃基质置于体积浓度为10%的戊二醛磷酸缓冲液中,室温放置M小时,纯净水内超声清洗5分钟,真空干燥。 The glass substrate was placed with a U-shaped groove of a volume concentration of 10% glutaraldehyde phosphate buffer, incubated at room temperature M hours, purified water ultrasonic cleaning for 5 minutes, and dried in vacuo.

[0024] 3、玻璃基质U型槽内表面的核酸探针固定: [0024] 3, nucleic acid U-groove surface of the glass substrate probe immobilization:

体积为0. 1 μ L、摩尔浓度为10 μ Lmol/L生物素标记的核酸探针溶液经毛细作用吸入内径为250 μ m玻璃点样管内,加至玻璃基质U型槽内壁的靶分子标记位置,参见图2所示, 室温避光静默M小时,在质量浓度为0. 1%的十二烷基硫酸钠溶液中超声清洗5分钟,超纯水清洗3次,真空干燥30分钟,4°C保存。 A volume of 0. 1 μ L, molar concentration of 10 μ Lmol / L biotin-labeled nucleic acid probe solution was suction capillary action inner diameter 250 μ m spotted glass tube, were added to U-shaped groove of the inner wall of the glass substrate labeled target molecule position, see Fig. 2, M silent hours dark at room temperature, at the concentration of 0.1% sodium dodecyl sulfate solution was ultrasonically cleaned for 5 minutes, washed three times with ultrapure water, and dried under vacuum for 30 minutes, 4 ° C hold.

[0025] 4、高分子记忆材料套管清洗: [0025] 4, polymer memory material ferrule cleaning:

将内径为3. 0mm,热缩比为2 :1的铁氟龙透明高聚物热缩材料套管置于体积浓度为6. 5%的硝酸溶液中,超声清洗30分钟;再置于质量浓度为10%的氢氧化钠溶液中,超声清洗30分钟,超纯水清洗;100 μ L无水乙醇来回穿梭冲洗30分钟,超纯水清洗;真空干燥30 分钟;洁净工作区,室温保存。 The inner diameter of 3. 0mm, shrink ratio of 2: 1 of a transparent polymer Teflon heat shrink sleeve material is placed in a volume concentration of 6.5% nitric acid solution, 30 minutes ultrasonic washing; and placed in mass concentration of 10% sodium hydroxide solution, ultrasonically cleaned for 30 minutes with ultrapure water; 100 μ L of absolute ethanol and fro rinsed for 30 minutes with ultrapure water; 30 minutes was dried in vacuo; clean saved workspace, rt.

[0026] 5、一种热缩组合式微通道芯片的组合: [0026] 5. A combination of micro-channel chip shrink in combination:

经核酸探针固定的玻璃基质U槽放置于清洗后的热缩材料套管中,80°C烘箱内均勻受热5分钟,热缩后套管高度透明,与玻璃基质U槽结合紧密,参见图1。 Nucleic acid probe immobilized via U-shaped groove placed in the glass matrix material of the sleeve after the thermal cleaning, uniform heating within the oven 80 ° C for 5 minutes and highly transparent heat-shrinkable tubing, in conjunction with the glass substrate tight U-groove, see FIG. 1.

[0027] 本发明所涉及的热一种热缩组合式微通道芯片的应用方法如下: 1、微通道芯片预杂交: [0027] The application of the method one kind of heat shrink composition of the micro-channel chip according to the present invention is as follows: 1, micro-channel chip pre-hybridization:

芯片腔道内吸入15 μ L的预杂交液,预杂交液成份为:100μ g/ml鲑鱼精、质量浓度为0. 1%的十二烷基硫酸钠溶液、质量浓度为0. 1%的牛血清白蛋白和5X柠檬酸钠缓冲液,微通道芯片垂直固定于杂交仪内旋转杆上,在42°C、40转/分钟的条件下,预杂交液在微通道芯片腔道内穿梭20分钟,可适当调整旋转速度以保证预杂交液能穿梭腔道全长。 Chip suction lumen 15 μ L of pre-hybridization solution, prehybridization solution ingredients: 100μ g / ml salmon sperm, at a concentration of 0.1% by mass of sodium lauryl sulfate solution at the concentration of 0.1% bovine serum albumin and sodium citrate buffer 5X, micro-channel chip perpendicularly fixed to the rotating rod within the hybridizer at 42 ° C, 40 revolutions / minute, in a prehybridization solution shuttle micro-channel chip cavity for 20 minutes, the rotational speed can be adjusted to ensure that the pre-hybridization solution can be full-length shuttle cavity. IOml超纯水以30ml/min流速冲洗微通道芯片腔道,IOOOrpm水平式离心3分钟。 IOml ultrapure water / min flow rate 30ml rinse micro-channel chip cavity, IOOOrpm horizontal centrifuge for 3 minutes.

[0028] 2.脱氧核苷酸序列靶分子与杂交溶液按1 :4体积比混合得到杂交反应液,杂交溶液的成份为:体积浓度为50%的去离子甲酰胺、5X柠檬酸钠缓冲液和质量浓度为0. 1% 的十二烷基硫酸钠溶液,杂交仪内,42°C、15 μ L杂交反应液、40转/分钟的条件下,在微通道芯片腔道内穿梭30分钟,可适当调整旋转速度以保证杂交反应液能穿梭腔道全长。 [0028] 2-deoxy nucleotide sequence of the target molecule and the hybridization solution 1: 4 volume ratio to obtain a reaction solution hybridization, the hybridization solution composition is: a volume concentration of 50% deionized formamide, 5X sodium citrate buffer under conditions and at a concentration of 0.1% by mass of sodium lauryl sulfate solution, the hybridizer, 42 ° C, 15 μ L hybridization reaction solution, 40 rev / min, the shuttle within the micro-channel chip cavity 30 minutes. the rotational speed can be adjusted to ensure that the hybridization reaction solution can be the entire length of the shuttle cavity.

[0029] 3.杂交混合液洗脱: [0029] 3. Elution hybridization cocktail:

55°C条件下,将5. Oml洗脱液1以lOml/min流速冲洗微通道芯片腔道,100 μ L洗脱液1的成份为:20Χ柠檬酸钠缓冲液10 μ L、质量浓度为10%的十二烷基硫酸钠溶液1 μ L和无菌超纯水89 μ L ;然后将IOml洗脱液2以lOml/min流速冲洗微通道芯片腔道,100 μ L洗脱液2的成份为:20Χ柠檬酸钠缓冲液0. 5 μ L、质量浓度为10%的十二烷基硫酸钠溶液1 μ L和无菌超纯水98. 5 μ L。 55 ° C for conditions 1 to 5. Oml eluent to lOml / min flow rate of irrigation micro-channel chip cavity, 100 μ L of eluent composition 1: 20Χ sodium citrate buffer, 10 μ L, the concentration of 10% sodium lauryl sulfate solution and 1 μ L of sterile ultrapure water to 89 μ L; 2 eluate was then IOml at lOml / min flow rate of irrigation micro-channel chip cavity, 100 μ L of eluent 2 ingredients: 20Χ sodium citrate buffer, 0. 5 μ L, concentration of 10% by mass sodium lauryl sulfate solution and 1 μ L of sterile ultrapure water 98. 5 μ L.

[0030] 4.亲和链霉素-碱性磷酸酶与核酸靶序列连接: [0030] 4. Affinity streptomycin - alkaline phosphatase and the nucleic acid target sequence is connected:

芯片腔道内吸满由ρΗ为7. O的磷酸盐缓冲液经1500倍稀释后的亲和链霉素-碱性磷酸酶,于37°C下避光放置20分钟,再由10mL、ρΗ为7. O的磷酸盐缓冲液以lOml/min流速冲洗芯片腔道。 Suck diluted to 1500-fold by the ρΗ 7. O phosphate buffer and affinity-streptomycin chip cavity - alkaline phosphatase, placed in a dark 37 ° C and 20 minutes, then to a 10mL, ρΗ 7. O phosphate buffer cavity to flush chips lOml / min flow rate.

[0031] 5.化学显色: [0031] 5. A Color:

将25 μ L显色液加入微通道芯片腔道,37°C下避光放置20分钟,显色液的成分为: 5-溴-4-氯-3-吲哚基-磷酸盐、氯化硝基四氮唑蓝和磷酸盐缓冲液,质量比为5 :10 : 1500。 The color developing solution 25 μ L was added micro-channel chip cavity, 37 ° C under the dark for 20 minutes, the color component solution is: 5-Bromo-4-chloro-3-indolyl - phosphate, chloride and nitro blue tetrazolium in phosphate buffer, mass ratio of 5: 10: 1500.

[0032] 6.结果观察: [0032] 6. Results observed:

拍照记录结果。 Photographs record the results.

[0033] 参见图3,检测结果表明:阳性结果明显,阴性背景色低;1号样品检测为野性基因型;2号样品检测为野生、突变混合基因型;3号样品检测为突变基因型。 [0033] Referring to FIG 3, test results showed that: significant positive result, a negative low background color; wild detected as sample No. 1 genotype; Sample No. 2 is detected as the wild-mutant genotype mixed; Sample No. 3 detects a mutant genotype.

Claims (10)

1.热缩组合式微通道芯片的制备方法,其特征在于: 由以下步骤实现:步骤一:将带有U型槽的玻璃基质置于体积浓度为6. 5%酸溶液中,70°C水浴超声清洗1小时;再置于质量浓度为10%的碱溶液中,80°C水浴超声清洗1小时;超纯水清洗后,真空干燥30分钟;步骤二:对玻璃基质的U型槽内壁进行氨基化处理和醛基化处理; 步骤三:体积为0. 1 μ L、摩尔浓度为10 μ mol/L生物素标记的核酸探针溶液经毛细作用吸入内径为250 μ m玻璃点样管内,加至玻璃基质的U型槽内壁,室温避光静置M小时, 在质量浓度为0. 1%的十二烷基硫酸钠溶液中超声清洗5分钟,超纯水清洗3次,真空干燥30分钟,4°C保存;步骤四:将内径为3. 0 mm,热缩比为2 :1的铁氟龙透明高聚物热缩材料套管置于体积浓度为6. 5%的酸溶液中,超声清洗30分钟;再置于质量浓度为10%的碱溶液中,超声清洗30分 A method for producing micro-channel chip thermal composition, wherein: implemented by the following steps: Step one: The glass substrate was placed with a U-shaped groove 6.5% volume concentration of the acid solution, 70 ° C water bath ultrasonic cleaning one hour; then placed in an alkali solution concentration of 10% by mass of, 80 ° C water bath for 1 hour ultrasonic cleaning; washing ultrapure water, and dried under vacuum for 30 min; step two: the inner wall of the U-groove substrate is glass amino and aldehyde treatment process; step three: a volume of 0. 1 μ L, molar concentration of the nucleic acid probe 10 μ mol / L biotin-labeled capillary action inhalation solution was 250 μ m inner diameter of the glass tube spotting, U-shaped groove of the inner wall applied to the glass substrate, allowed to stand at room temperature in the dark M h, at the concentration of 0.1% sodium dodecyl sulfate solution was ultrasonically cleaned for 5 minutes, washed three times with ultrapure water, and dried in vacuo 30 min, 4 ° C preservation; step four: an inner diameter of 3. 0 mm, heat shrink ratio of 2: 1 of a transparent polymer Teflon heat shrink sleeve material disposed concentration of 6.5% by volume of the acid solution , the ultrasonic cleaning 30 min; then placed in an alkali solution concentration of 10% by mass, the ultrasonic cleaning for 30 minutes 钟,超纯水清洗;100 μ L无水乙醇来回穿梭冲洗30分钟,超纯水清洗;真空冷冻干燥, 洁净工作区保存,室温保存;步骤五:将U型槽内固定有核酸探针的玻璃基质放置于清洗后的热缩材料套管中, 80°C真空干燥箱内均勻受热5分钟,热缩后套管高度透明,与玻璃基质的U型槽结合紧密。 Clock, and ultrapure water; 100 μ L of absolute ethanol and fro rinsed for 30 minutes with ultrapure water; vacuum freeze dry, clean work areas, stored at room temperature; Step 5: U-shaped groove of the nucleic acid probe fixed glass substrate is placed in heat-shrinkable sleeve material after washing, heat evenly within 80 ° C vacuum oven for 5 minutes and highly transparent heat-shrinkable tubing, in conjunction with the U-groove substrate glass tightly.
2.根据权利要求1所述的热缩组合式微通道芯片的制备方法,其特征在于:步骤一中的酸溶液为硝酸溶液,碱溶液为氢氧化钠溶液。 The production method of claim 1 in combination thermal micro-channel chip according to claim wherein: a step in a solution of nitric acid solution, an alkaline solution is sodium hydroxide solution.
3.根据权利要求1所述的热缩组合式微通道芯片的制备方法,其特征在于:步骤二中的氨基化处理由以下步骤实现:经步聚一处理后的带有U型槽的玻璃基质浸泡于体积比为1 :9的3-氨基丙基三甲氧基硅烷/甲苯溶液中,置于80°C水浴超声30分钟, 重复4次,1000转/分钟水平式离心5分钟;依次用分析纯甲苯溶液、体积浓度为95%的乙醇溶液超声10分钟;步骤二中的醛基化处理由以下步骤实现:将经过氨基化处理过的带有U型槽的玻璃基质置于体积浓度为10%的戊二醛磷酸缓冲液中,室温放置M小时,纯净水内超声清洗5分钟,真空干燥。 The method for preparing a heat shrinkable composition according to claim micro-channel chip, wherein: the step of amination of the second processing is realized by the following steps: the glass substrate by Buju treated with a U-shaped groove immersed in a volume ratio of 1: 3-aminopropyl trimethoxysilane / toluene solution 9, placed in an ultrasonic water bath at 80 ° C for 30 minutes was repeated four times, 1000 rev / min horizontal centrifuged for 5 minutes; washed with analysis pure ethanol solution was sonicated in toluene, concentration of 95% by volume 10 min; step two aldehyde treatment is implemented by the steps of: after amination treated glass substrate was placed with a U-shaped groove volume concentration of 10 % glutaraldehyde phosphate buffer, M h incubated at room temperature, the pure water ultrasonic cleaning for 5 minutes, and dried in vacuo.
4.根据权利要求1所述的热缩组合式微通道芯片的制备方法,其特征在于:步骤四中的酸溶液为硝酸溶液,碱溶液为氢氧化钠溶液。 The compositions shrink microchannels chip production method according to claim 1, wherein: in step four acid solution is a solution of nitric acid, an alkali solution is sodium hydroxide solution.
5.如权利要求1所述的制备方法得到的热缩组合式微通道芯片。 5. The micro-channel chip shrink composition production method according to claim 1 obtained.
6.热缩组合式微通道芯片的应用方法,其特征在于: 由以下步骤实现:步骤一:芯片腔道内吸入15 μ L的预杂交液,热缩组合式微通道芯片垂直固定于杂交仪内旋转杆上,在42°C、40转/分钟的条件下,预杂交液在微通道芯片腔道内穿梭20分钟, IOml超纯水以30ml/min流速冲洗微通道芯片腔道,1000转/分钟水平式离心3分钟;步骤二:脱氧核苷酸序列靶分子与杂交溶液按1 :4体积比混合得到杂交反应液,杂交仪内,42Ό、15μ L杂交反应液、40转/分钟的条件下,在微通道芯片腔道内穿梭30分钟;步骤三:55°C条件下,将5. Oml洗脱液1以lOml/min流速冲洗微通道芯片腔道,然后将IOml洗脱液2以lOml/min流速冲洗微通道芯片腔道;步骤四:芯片腔道内吸满由PH为7.0的磷酸盐缓冲液经1500倍稀释后的亲和链霉素-碱性磷酸酶,于37°C下避光放置20分钟,再由IOml、pH为7. 0的磷酸盐缓冲液 6. The method of application of a combination of thermal micro-channel chip, wherein: implemented by the following steps: Step 1: the chip suction cavity 15 μ L of the prehybridization solution, a combination of micro-channel chip shrink perpendicularly fixed to the rotating lever hybridizer under conditions at 42 ° C, 40 revolutions / minute, the pre-hybridization solution running through a micro-channel chip cavity 20 minutes, IOml ultrapure water / min flow rate 30ml rinse micro-channel chip cavity 1000 rev / min horizontal centrifuged for 3 min; step two: a deoxynucleotide with the sequence of the target molecule hybridization solution of 1: 4 to obtain the volume ratio of the hybridization reaction solution, the lower the hybridizer, 42Ό, 15μ L hybridization reaction solution, 40 revolutions / minute, in the micro-channel chip cavity shuttle 30 minutes; three steps: 55 ° C for conditions 1 to 5. Oml eluent to lOml / min flow rate of irrigation micro-channel chip cavity, and then the eluate IOml 2 to lOml / min flow rate rinse micro-channel chip cavity; step four: suck the chip cavity PH 1500-fold dilution of the phosphate buffer was 7.0 affinity streptomycin - alkaline phosphatase, dark place at 37 ° C for 20 minutes, then the IOml, pH 7.0 phosphate buffer solution of 以IOml/ min流速冲洗芯片腔道;步骤五:将25 μ L显色液加入微通道芯片腔道,37°C下避光放置20分钟;步骤六:观察实验结果并拍照记录。 In IOml / min flow rate of flush chip cavity; Step 5: 25 μ L were added to a color micro-channel chip cavity, 37 ° C protected from light for 20 min; Step Six: The results were observed and photographed record.
7.根据权利要求6所述的热缩组合式微通道芯片的应用方法,其特征在于:所述的步骤一中的预杂交液成份为lOOug/ml鲑鱼精、质量浓度为0. 1%的十二烷基硫酸钠溶液、质量浓度为0. 1%的牛血清白蛋白和5X柠檬酸钠缓冲液。 7. The method of claim 6 Application of micro-channel chip shrink composition according to claim wherein: said step of pre-hybridization in a liquid component of lOOug / ml salmon sperm, at a concentration of 0.1% by mass of the ten sodium lauryl sulfate solution at the concentration of 0.1% bovine serum albumin and sodium citrate buffer 5X.
8.根据权利要求6所述的热缩组合式微通道芯片的应用方法,其特征在于:所述的步骤二中的杂交溶液的成份为体积浓度为50%的去离子甲酰胺、5X柠檬酸钠缓冲液和质量浓度为0. 1%的十二烷基硫酸钠溶液。 8. The method of claim 6 use a combination of micro-channel chip shrink claim, wherein: the hybridization solution components in step two of the volume concentration of 50% deionized formamide, 5X sodium citrate buffer and the concentration of 0.1% sodium dodecyl sulfate solution.
9.根据权利要求6所述的热缩组合式微通道芯片的应用方法,其特征在于:所述的步骤三中的洗脱液l,100yL体积的成份为20X柠檬酸钠缓冲液10 μ L、质量浓度为10%的十二烷基硫酸钠溶液1 μ L和无菌超纯水89 μ L ;所述的洗脱液2,100 μ L体积的成份为20 X柠檬酸钠缓冲液0.5 μ L、质量浓度为10%的十二烷基硫酸钠溶液Iul和无菌超纯水98. 5μ L。 9. The method of claim 6 Application of micro-channel chip shrink composition according to claim wherein: in step three of the eluate l, 100yL 20X volume of ingredients for sodium citrate buffer 10 μ L, mass concentration of 10% solution of sodium dodecyl sulfate and 1 μ L of sterile ultrapure water to 89 μ L; said volume of eluate 2,100 μ L ingredients for 20 X 0.5 μ sodium citrate buffer sodium lauryl sulfate solution Iul L, concentration of 10% by mass and sterile ultra-pure water 98. 5μ L.
10.根据权利要求6所述的一种热缩组合式微通道芯片的应用方法,其特征在于:所述的步骤五中的显色液的成分为5-溴-4-氯-3-吲哚基-磷酸盐、氯化硝基四氮唑蓝和磷酸盐缓冲液,体积比为5 :10 :1500. A method for application of micro-channel chip shrink 10. A composition according to claim 6, wherein: said component of the fifth step of color developing solution is 5-bromo-4-chloro-3-indolyl group - phosphate, nitro blue tetrazolium chloride and phosphate buffer, the volume ratio of 5: 10: 1500.
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